Continuous-labeling HDX-MS on spike 2P. HDX-MS offers an ideal complement to the ever-growing number of structural studies on the SARS-CoV-2 spike protein, providing information on its conformational ensemble and dynamics. HDX-MS monitors the time course of the exchange of amide hydrogens on the peptide

Outer membrane protein (OMP) biogenesis is critical to bacterial physiology because the cellular envelope is vital to bacterial pathogenesis and antibiotic resistance. The process of OMP biogenesis has been studied in vivo, and each of its components has been studied in isolation in vitro. This work integrates parameters and observations from both in vivo and in vitro experiments into a holistic computational model termed "Outer Membrane Protein Biogenesis Model" (OMPBioM). We use OMPBioM to assess OMP biogenesis mathematically in a global manner. Using deterministic and stochastic methods, we are able to simulate OMP biogenesis under varying genetic conditions, each of which successfully replicates experimental observations. We observe that OMPs have a prolonged lifetime in the periplasm where an unfolded OMP makes, on average, hundreds of short-lived interactions with chaperones before folding into its native state. We find that some periplasmic chaperones function primarily as quality-control factors; this function complements the folding catalysis function of other chaperones. Additionally, the effective rate for the β-barrel assembly machinery complex necessary for physiological folding was found to be higher than has currently been observed in vitro. Overall, we find a finely tuned balance between thermodynamic and kinetic parameters maximizes OMP folding flux and minimizes aggregation and unnecessary degradation. In sum, OMPBioM provides a global view of OMP biogenesis that yields unique insights into this essential pathway.T he cellular envelope of Gram-negative bacteria is comprised of two membranes separated by an aqueous compartment termed the "periplasm." The outer membrane of the cellular envelope contains integral β-barrel membrane proteins referred to as "outer membrane proteins" (OMPs) (1, 2). The outer membrane and OMPs provide the first barrier between bacteria and the environment and are essential to many important cellular processes including metabolic transport, bacterial virulence, and antibiotic resistance (3-5). Understanding the pathway by which OMPs traverse the periplasm and attain their native functional state is essential to an ability to manipulate this element of the bacterial cell.The OMP biogenesis process is distinct from the folding of cytosolic proteins because it involves a unique collection of obstacles. First, OMPs do not adopt their folded conformations while in an aqueous environment (6). Rather, unfolded OMPs (uOMPs) must be transported across the periplasm to reach their native membrane. Because of their marginal solubility in water, this process must be tightly controlled to avoid aggregation. Second, structures of folded OMPs (fOMPs) show that these proteins contain water-solvated residues in loops on the outer surfaces of bacteria. The desolvation and transport of these polar and ionizable side chains across the outer membrane represent significant kinetic barriers to OMP folding (7). Third, Gram-negative bacteria maintain a high density of fOMP in the expanding oute...

Kinetic and structural comparison of the cotranslational and refolding pathways of HaloTag reveals how translation reroutes the folding pathway to avoid aggregation.

To address the ongoing SARS-CoV-2 pandemic and prepare for future coronavirus outbreaks, understanding the protective potential of epitopes conserved across SARS-CoV-2 variants and coronavirus lineages is essential. We describe a highly conserved, conformational S2 domain epitope present only in the prefusion core of β-coronaviruses: SARS-CoV-2 S2 apex residues 980–1006 in the flexible hinge. Antibody RAY53 binds the native hinge in MERS-CoV and SARS-CoV-2 spikes on the surface of mammalian cells and mediates antibody-dependent cellular phagocytosis and cytotoxicity against SARS-CoV-2 spike in vitro. Hinge epitope mutations that ablate antibody binding compromise pseudovirus infectivity, but changes elsewhere that affect spike opening dynamics, including those found in Omicron BA.1, occlude the epitope and may evade pre-existing serum antibodies targeting the S2 core. This work defines a third class of S2 antibody while providing insights into the potency and limitations of S2 core epitope targeting.